Linking anatomical and histological traits of the digestive tract to resource consumption and assimilation of omnivorous tetra fishes

Abstract This study explores the interplay between digestive tract traits, food intake, and assimilation in omnivorous tetra fishes (Psalidodon bifasciatus, P. aff. gymnodontus, and Bryconamericus ikaa) from the Iguaçu River basin, an ecologically significant region known for high endemism. We hypothesize that variations in digestive tracts across species would be associated with differences in diet, isotopic composition in fish tissues, and overall diet assimilation. To test this, we employed stereoscopic and light microscopy to characterize the gross anatomy, histomorphology, and histochemistry of fish digestive tracts. Additionally, we used stomach content and stable isotope analyses to trace fish diets. While these tetra fishes shared histological structures, disparities were noted in anatomical digestive traits and diet preferences. The smallest species, B. ikaa, with a shorter intestine, had fewer pyloric caeca and primarily consumed animal‐based diets. Conversely, P. bifasciatus and P. aff. gymnodontus, with longer intestines, displayed numerous pyloric caeca and consumed a balanced mix of animal and plant items. Despite anatomical and dietary differences, all three species predominantly assimilated animal‐origin food. The tetra fishes had histological variations among digestive tract segments, with the esophagus having the thickest muscular layer, gradually thinning towards the posterior intestine. The final portion of the intestine exhibited a significant expansion in the lumen perimeter, while the esophagus had the smallest lumen area. Goblet cells were most concentrated in the posterior intestine for all species. The gross anatomy of these tetra fishes aligns with their omnivorous habit, while diet assimilation was dominated by animal‐origin food. These findings provide crucial insights into the structural and tissue characteristics of their digestive systems, laying the groundwork for deeper exploration into the physiological aspects of their digestive tracts and enhancing our understanding of their feeding strategies.


| INTRODUC TI ON
Resource acquisition is vital for the survival of species, with animals evolving to exploit various food types (Karasov et al., 2011).Wild fishes face the challenge of meeting their nutritional needs amidst variations in food availability and quality, possibly driving the development of diverse digestive adaptations.The variability in the digestive system appears to correlate with materials resistant to digestion (Karasov et al., 2011).Physical, chemical, and enzymatic processes contribute to digestion, while absorption and assimilation incorporate nutrients into cells and tissues for various physiological functions (Bakke et al., 2010;Nielsen et al., 2018).Consequently, understanding the morphological traits of digestive systems is crucial for comprehending the relationship between ingestion, digestion, absorption, and assimilation of food, particularly in dynamic environments like tropical streams and rivers (Delariva & Neves, 2020;Neves et al., 2023;Wilson & Castro, 2010).
The morphology of the digestive tract in teleost fishes, essential for their diverse feeding strategies, has been extensively studied, revealing various anatomical adaptations and trophic position (Moraes & Almeida, 2020).However, our understanding of the diversity of teleost digestive systems remains limited due to the highly morphological and phylogenetic diversification (Albrecht et al., 2001;Dzhumaliyev, 1982;Fanta et al., 2001;Karasov et al., 2011).While histological relationships between diet and digestive tract have previously been documented, descriptive data, especially for small omnivorous Neotropical fishes, are still lacking (Alonso et al., 2015;Cardoso et al., 2015;Rincón et al., 2023).Teleosts exhibit morphological adaptations reflecting their varied diets, influencing the length of their intestines and nutrient acquisition efficiency (Ghilardi et al., 2021;Ribble & Smith, 1983;Wilson & Castro, 2010).Studies of digestive systems advance our understanding of fish physiology and feeding habits (Fugi & Hahn, 1991;Végaz-Velez, 1972), crucial for comprehending digestive performance and evolution, especially in species-rich yet phylogenetically correlated neotropical environments (Albert et al., 2020;Baumgartner et al., 2012).
The family Characidae is known for its remarkable diversity among fish taxa in the Neotropical freshwater ecosystems (Fricke et al., 2020).The small characids, commonly referred to as tetra fish, are particularly abundant in rivers, lakes, and streams.Psalidodon, Astyanax, and Bryconamericus are the most species-rich genera of the Characidae family.Omnivorous diet and trophic plasticity have been well documented for these genera that often consume both animal and plant food items (Bonato et al., 2018;Neves et al., 2023;Pini et al., 2019).These species are often opportunistic feeders with the ability to change their diet across seasons and space depending on food resource availability (Neves et al., 2021(Neves et al., , 2023;;Pini et al., 2019;Quirino et al., 2015).Although the trophic ecology of these species has been extensively studied (Bonato et al., 2018;Delariva & Neves, 2020;Neves et al., 2023;Pini et al., 2019), we still know little about the histological characteristics of their digestive tract and how these traits are related to food ingestion, digestion, absorption, and assimilation.Moreover, recent stable isotope analyses indicate that while tetra fishes readily ingest plant food items, they rarely assimilate or incorporate these types of food into their tissues (Bonato et al., 2018;Neves et al., 2021Neves et al., , 2023)).This finding raises questions about the role of plant material in the diets of tetra fishes.
In this study, we explored the relationships among morphological characteristics of the digestive tract, food intake, isotopic composition, and resource assimilation of three native omnivorous fish spe-  (Neves et al., 2023).Consequently, our study aimed to accomplish the following objectives: (i) describe the anatomical, histological, and histochemical traits of the digestive tract and diet of these small tetra fishes; (ii) investigate the potential relationship between intestine length, diet, and isotopic composition given their omnivorous feeding habit; and (iii) test the hypothesis that variations in the digestive tract among the species correspond to differences in resource utilization (animal and plant food items), isotopic composition, and assimilation.Such evidence would advance our understanding of morphology, trophic plasticity, and species coexistence among fish species in the Iguassu Ecoregion, offering crucial ecological insights.Understanding these relationships is essential to improve conservation efforts, ensuring the preservation of the unique biodiversity and ecological integrity of this ecoregion.

| Species acquisition
To collect the tetra fishes, we sampled two headwater streams (S1: Ecophysiology, Functional ecology, Trophic interactions, Zoology specimens according to specific identification keys (Baumgartner et al., 2012;Ota et al., 2018), measured the total and standard length from the left side of the individuals using a digital caliper (accuracy of 0.01 mm), and weight (g) using a portable digital scale (Vazzoler, 1996).We then visually inspected the presence of mature gonads of all sampled fishes and selected adult specimens of each species for further analysis (Vazzoler, 1996).
For the histological analysis, during each sampling period, we selected five individuals of each species from each stream.We dissected their digestive tracts with a longitudinal incision along the ventral region.Then, we fixed the digestive tract in ALFAC (alcohol 80%, 85 mL; formaldehyde PA, 10 mL; and acetic acid PA, 5 mL; Caputo et al., 2011) and conserved in 70% alcohol.We also fixed additional specimens in formaldehyde 10% and conserved them in 70% alcohol for stomach content analysis.Finally, for stable isotope analysis, in the summer (December 2017), we dissected samples of dorsal muscle tissue from 10 to 15 adult specimens per species and stream.Following the methodology from Neves et al. (2021Neves et al. ( , 2023)), we manually sampled basal resources (terrestrial plants and sedimentary organic matter-SOM) and putative prey (aquatic and terrestrial invertebrates).We immediately stored all samples on ice for further processing in the laboratory.
We collected the fishes under authorization from the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio) (license number 25039).The project was ethically and methodologically approved by the Ethics Committee on Animal Use of the Universidade Federal do Rio Grande do Sul (CEUA -32734).To comply with their protocols for fish usage, we deposited the voucher specimens in the fish collection of the Departamento de Zoologia at the Universidade Federal do Rio Grande do Sul (P.bifasciatus UFRGS 26235; P. aff.gymnodontus UFRGS 25725; B. ikaa UFRGS 26246).

| Stomach content analysis
To estimate stomach contents, we analyzed 244 adult fish specimens (B.ikaa: 31; P. aff.gymnodontus: 96; P. bifasciatus: 117).We carefully extracted the stomachs of the fishes under optical and stereoscopic microscopes (Opton TIM-2B WF10X) to identify the stomach contents with the highest possible taxonomic precision.
For the identification of algae we used specialized literature by Bicudo and Bicudo (1970), while for invertebrates, we used Mugnai et al. (2010).We applied the volumetric method to quantify the food items (Hyslop, 1980) with graduated test tubes and glass counting plates (Hellawell & Abel, 1971).
Muscle samples were first washed with distilled water, then lyophilized and homogenized with a mortar and pestle.We then stored all samples in 2 mL Eppendorf tubes until they were weighed into tin capsules, with each capsule containing approximately 1.6 ± 0.2 mg of dried animal tissue.Similarly, we washed all basal resources and prey items with distilled water.We then lyophilized and homogenized aquatic and terrestrial invertebrates (separated by taxonomic groups), and basal resources (Table S7).The small aquatic insects, such as Ephemeroptera, Chironomidae, Coleoptera, and Hymnoptera, underwent complete maceration.Larger prey items, including shrimps and crabs (Aegla sp.) were freeze-dried and their muscle tissue was macerated.We preserved all specimens in 2 mL Eppendorf tubes before measuring and transferring them into tin capsules (1.6 ± 0.2 mg of dry animal tissue and 3.6 ± 4.2 mg of basal resources).We carried out the analysis of nitrogen ( 15 N/ 14 N) and carbon ( 13 C/ 12 C) stable isotope ratios at the Center for Nuclear Energy in Agriculture (CENA) at the University of São Paulo, Brazil.To determine the stable isotope ratios, CENA used a mass spectrometer system operating in continuous-flow (CF-IRMS) mode.This system was fit with a Carlo Erba elemental analyzer (CHN 1110) connected to a Delta Plus mass spectrometer (Thermo Scientific).The results of the stable isotope analysis were presented in the delta notation, which represents the deviation of stable isotope ratios ( 13 C: 12 C and 15 N: 14 N) from universal standards.Specifically, the carbon ratios were compared to the PDB limestone standard, while the nitrogen ratios were compared to atmospheric nitrogen.For the analysis of fish muscle δ 13 C, we did not correct lipids due to the low C:N ratios observed (below 3.5), indicating negligible lipid content in the samples (Hoffman et al., 2015).

| Gross anatomy
To investigate the gross morphology of the digestive tract of tetra fishes, we measured the intestinal length with digital calipers (accuracy of 0.01 mm) and counted the number of pyloric caeca of a total of 55 adult fish specimens (B.ikaa: 10; P. aff.gymnodontus: 15; P. bifasciatus: 30).We computed the intestinal coefficient (IC) using the Hynes (1950) model, following the formula IC = IL/SL, where IL denotes the length of the intestine in mm, while SL denotes the standard length, also measured in mm.We photographed anatomical and macroscopic characteristics of the digestive tract of species using a Multipurpose Zoom Microscope Nikon AZ100M.

| Histology and histochemistry
To describe the microscopic anatomy and histology of the digestive tract of tetra fishes, we examined a total of 40 specimens (B.ikaa: 10; P. aff.gymnodontus: 10; P. bifasciatus: 20).We used the ALFACfixed material and selected tissue fragments from the esophagus, stomach, and three portions of the intestine (anterior, middle, and posterior; Figure 1) of species and washed with 70% ethanol to remove the food items.These tissue fragments were then dehydrated in graded ethanol solutions and embedded in historesin (Leica®).We then made transverse and longitudinal histological sections (2-3 μm) with a Leica microtome (model RM 2145).Next, we stained the histological sections with 1% toluidine blue (TB), periodic acid Schiff (PAS) with alcian blue (AB -pH 2.5), and Masson's trichrome staining (MT).We used PAS + AB to detect acid and neutral mucins (Cao & Wang, 2009).Lastly, we used Masson's trichrome staining to better visualize the collagen fibers.We observed all sections using a BX60 Olympus microscope and recorded the images using an Olympus DP71 digital camera and DP Controller 3.2.1.276software.
For a quantitative examination of the digestive tract traits, we took three histological photographs of each segment from five individuals per species.Following the methodology outlined by Bellinate et al. (2023) and Curvo et al. (2020), we used histological photographs and Fiji-ImageJ software (Schindelin et al., 2012) to determine the number of goblet cells, thickness of the muscle layer, and the perimeter and area of the lumen, as a proxy for the absorption surface.We analyzed these variables only for the esophagus and intestine segments, as the degree of stomach fullness (i.e., full vs. empty) could cause measurement bias.Because goblet cells completely made up the esophageal epithelium, we focused on accounting for this cell type in the anterior, middle, and final segments of the intestine.We counted the number of goblet cells by randomly choosing four microvilli in each segment of the intestine.We standardized the measurements in micrometers using the scales of the histological photographs.

| Statistical analyses
To assess any differences in the consumption of animal-and plantbased food items among different species, we categorized the food items into three distinct food groups: (i) animal origin, including both aquatic and terrestrial invertebrates; (ii) plant origin, encompassing seeds and plant remnants; and (iii) undetermined origin, which consisted of detritus at various stages of decomposition and mineral particles.Subsequently, we performed a one-way PERMANOVA analysis on an individual fish food item matrix, employing the Bray-Curtis index with 9999 permutations, as outlined by Anderson (2001).
Tissue stable isotopes can be used as tracers to determine food assimilation by different species (Nielsen et al., 2018).Nitrogen stable isotope ratios (δ 15 N) allow to estimate the trophic positions of organisms, whereas carbon stable isotope ratios (δ 13 C) allow to identify carbon sources of the diet items for aquatic consumers.
Because δ 13 C changes very little with assimilation of food sources into consumers, it serves as an indicator of carbon sources, especially distinguishing between C3 and C4 plants or between aquatic and terrestrial food items.On the other hand, δ 15 N undergoes trophic fractionation, increasing by 2%-4‰ with each trophic level, allowing the determination of consumer's trophic position and food chain length (Nielsen et al., 2018).Thus, to investigate the relationships between consumption (stomach content analysis), stable isotopic composition (δ 15 N and δ 13 C values), and intestinal coefficients (numerical variable) of tetra fishes (categorical variable), we used generalized linear mixed effects models (GLMMs) that assume a normal distribution in the variation of food resources (animal and plant resources), δ 15 N and δ 13 C values.We considered the streams as a random effect, while ICs and species were treated as fixed effects.
We fit the GLMMs using the lme4 package in R programming language (Bates et al., 2015).
In addition, to determine the relative contributions of different diet sources assimilated by fishes in each site, we applied Bayesian stable isotope mixing models (Parnell et al., 2010) et al., 2018;Phillips et al., 2014).Here, we considered three isotopically distinct food categories: (i) animal food items, (ii) plant food items, and (iii) SOM.We used trophic discrimination factors (TDF) of 1.3 ± 0.3% for C, and 2.9 ± 0.32% for N (McCutchan et al., 2003).This enrichment is appropriate for muscle tissues of omnivorous fishes that consume mixtures of plant and animal diet (McCutchan et al., 2003).We also considered specific TDF values for plant food items (Bastos et al., 2017).Because these fishes also assimilate bacteria and other microbes inhabiting basal plant resources, we followed the method of Neres-Lima et al. ( 2016 To examine potential differences whether the number of pyloric caeca differs among species, we conducted a one-way ANOVA, followed by the Tukey-HSD post hoc test.Regarding to the IC, considering possible differences in the standard length (SL) among species, we performed the analysis of covariance (ANCOVA) to assess whether the relationship between IC and species was influenced by the fish size.For these analyses, we used the vegan package (version 2.4-6) developed by Oksanen et al. (2022) and the car package (Fox & Weisberg, 2019).Similarly, to test whether there are differences in the thickness of the muscular layer, perimeter and lumen area, and number of goblet cells among segments of the digestive tract and species, we applied a two-way ANOVA, followed by the Tukey-HSD post hoc test.Before conducting the tests, we confirmed that the data met the assumptions of normality and homoscedasticity.All statistical analyses were performed in the language environment R, version 4.2.1 (R Core Team, 2022).

| Diet and isotopic composition
In agreement with our a-priory hypothesis, there were significant differences in the food use by the three tetra fishes (Pseud-F = 7.83, p < .001).Stomach content analysis revealed that the animal food items contributed the largest proportion to diet of B. ikaa (82.6%).By contrast, both P. bifasciatus and P. aff.gymnodontus consumed similar proportions of animal and plant food items (Figure 2).However, in contrast to stomach content analysis, there was no difference in mean δ 15 N and δ 13 C values among the three tetra species (Table 1).

| Gross anatomy
Tetra fishes had a compact and cylindrical esophagus, which led to a siphonal stomach characterized by a J-shaped saccular structure.
The stomach consists of three distinct regions, namely the cardiac, fundic, and pyloric regions (Figures 3-5).Additionally, the intestinal tract consisted of four primary loops.We observed pyloric caeca in the anterior portion of the intestine, and the number of pyloric caeca differed significantly among species (F 2,127 = 357.3;p < .0001, Figure 6).Similarly, there were significant differences in IC among species (F 2,124 = 507.1;p < .0001).However, there was a significant interaction between species and size (SL, F 2,124 = 5.10; p = .007),suggesting that differences in IC were associated to species size (Figure 6a).Specifically, B. ikaa, the smallest species, had a smaller IC and fewer pyloric caeca (8) than the other two species.
In contrast, P. bifasciatus had a longer intestine than other species (Figure 6).Both P. bifasciatus and P. aff.gymnodontus, the biggest species, had a similar number of pyloric caeca (10 or 11).

| Relationships among intestinal coefficients, consumption, and isotopic composition
As expected, there was a significant relationship between the consumption of animal food items and the IC among species (Table 1), indicating that the species with shorter intestine lengths, like B. ikaa, had more animals in their diet than the other two species.However, there was no significant relationship between isotopic composition and IC (Table 1), suggesting that species preferentially assimilated similar food types (i.e., animal origin) into their muscle tissues.

| Histology and histochemistry characterization
The digestive tracts of studied fishes had a similar histological composition, characterized by four distinct layers: mucosa, submucosa, muscular, and serosa.The esophageal epithelium displayed a simple squamous epithelium (Figure 7) and contained numerous mucous secreting cells (MSC) concentrated in the apical region and secreted both acid and neutral mucins (Figure 8).The submucosa layer was notably thick and comprised of dense connective tissue without any glands.This layer, greenish tones with Masson's trichrome staining (Figure 8a), had collagen fibers that provide mechanical strength.Lastly, the muscular tissue, dark red tones with TA B L E 1 The relationships between the intestinal coefficient (IC), consumption of animal and plant resources, and isotopic composition of tetra fishes obtained from generalized linear mixed effects models.Masson's trichrome staining (Figure 8a), consists of circular and longitudinal muscular layers.
The cardiac region of the stomach had well-defined long folds in all three species (Figure 9).The mucosa comprised a single layer of gastric epithelium with columnar epithelial cells (Figure 9a-c), with apical mucosubstances PAS+ (Figure 10).The submucosa contained many gastric glands (Figures 9 and 10), followed by an inner circular and an outer longitudinal muscular.The fundic region had a similar structure, but the gastric gland layer gradually reduced (Figure 8d-f).
Histological observations highlighted structural differences between the pyloric, cardiac, and fundic regions (Figure 9g-i).In the pyloric region, the primary variations observed were attributed to the lack of gastric glands and the existence of a substantial circular inner layer within the muscular layer (Figures 9g-i and 10c,f).The epithelium consisted of a single layer of columnar cells, which displayed positive staining for PAS+ in their apical region (Figure 9f).
The intestinal wall of the tetra fishes had a slender and extensively folded structure, except for the middle section in B. ikaa (Figure 11).The mucosa layer comprised a single layer of columnar epithelial cells known as enterocytes with a well-developed brush border.Additionally, goblet cells were present within the mucosa layer (Figure 12d-f).
Significant differences in the histological characteristics were observed mainly among the segments of the digestive tract (Tables S1   and S2).The thickness of the muscular layer differed significantly among segments of the digestive tract (F 3,48 : 546.4; p < .0001),with a significant interaction among species and segments (Table S2).
The muscular layer was significantly thicker in the esophagus, reduced in the anterior and middle segments of the intestine, and increased in the posterior intestine (Tables S1-S3).Lumen perimeter differed both among segments (F 3,48 : 7.51; p < .0001)and species (F 2,48 : 17.67; p < .0001).Specifically, the lumen perimeter tended to be greater in the final portion of the intestine and greater for P.

| DISCUSS ION
The studied tetra fish species differ in their proportions of animal-plant consumption yet share similar food assimilation patterns identified through stable isotope analyses.Despite differences in their digestive tract anatomy, these species maintain comparable histological structures.The intestine segments show variations in thickness, absorption surface, and goblet cell abundance.For instance, the B. ikaa, which is smaller in size and has shorter intestines, primarily feeds on animal-based diets, and has fewer pyloric caeca.
In contrast, larger Psalidodon species with longer intestines consume similar proportions of animal and plant foods and have more pyloric caeca.This suggests that despite belonging to the same family (Characidae), the evolutionary trajectories of the two genera have resulted in morphological and ecological differences that facilitated their dynamism of natural coexistence.These traits are closely related to the dietary plasticity and ecological success of each species within its environment.Therefore, the anatomy and functions of the digestive tract in these species are shaped by evolutionary, environmental, and ecological factors (Karasov et al., 2011).
In our study, B. ikaa had the lowest number of pyloric caeca and an aquatic insectivorous feeding habit.Psalidodon species, characterized as omnivorous, had longer intestines and numerous pyloric caeca, consuming insects, plants, and seeds.However, despite the morphological and dietary differences between these two genera, the main assimilated resource was of animal origin.Understanding why certain fish species consume plants without utilizing them for tissue building continues to be an exciting venue for future research, promising to advance our understanding of trophic plasticity in widely distributed omnivores.
The disparity between food consumption and assimilation in tetra fishes may be governed by several mechanisms, including (i) variations in digestibility and nutritional quality (Bowen et al., 1995), (ii) the lack of specialized adaptations in their digestive tracts for breaking down and assimilating plant material (Pelster et al., 2015), (iii) the ingested plant material may contain biofilm, a primary resource assimilated into fish tissues, and (iv) ecological and behavioral factors, such as opportunistic feeding strategies and accidental consumption (Bastos et al., 2017;Bonato et al., 2018).For P. bifasciatus and P. aff.gymnodontus, despite the consistent intake of plant material across seasons, the rates of assimilation remained low (Neves et al., 2021).While more comparative studies are necessary to establish the incongruence between plant intake and assimilation by fish, a plausible hypothesis, alongside the gastrointestinal microbiome (Karasov et al., 2011), suggests that all animals, irrespective of their diet, require protein (Gerking, 1994).Consequently, selection should not strongly favor animals with very low protein-processing capability (McWilliams, 2011).
Regarding the histological traits, the studied fishes had a general vertebrate pattern with the digestive tract wall (i.e., comprising four layers: mucosa, submucosa, muscular, and serosa).The esophagus and intestine shared similar histological features, featuring a mucosa with columnar epithelium and goblet cells.Notably, the muscular and submucosa layer showed a gradual thinning from the esophagus to the intestine.This submucosa layer, characterized by collagen fibers, serves a crucial function, imparting mechanical strength in the initial stages of the digestive tract, particularly in processes like swallowing (Moraes & Almeida, 2020).
The esophagus was characterized by a short and thick circular muscular layer, suggesting a specialized function for efficient food swallowing (Karasov et al., 2011).The presence of goblet cells in the esophageal epithelium potentially acts as a protective mechanism against damage caused by ingested food, since salivary glands are absent in fish (Faccioli et al., 2014;Scocco et al., 1998).Our analysis revealed the presence of acidic and neutral mucins in the esophagus, like other carnivorous and omnivorous fish species (Alonso et al., 2015;Faccioli et al., 2014;Germano et al., 2014).These mucins contribute to the thickness and stickiness of secretions, enhancing lubrication on epithelial surfaces to prevent mechanical damage and protect against potential pathogens (Abaurrea-Equisoaín & Ostos-Garrido, 1996;Fletcher & Grant, 1969;Humbert et al., 1984), beside immunological defense (Matheus et al., 2021).The presence of neutral mucins also indicates a possible pre-gastric digestion process through food emulsification (Cao & Wang, 2009;Murray et al., 1996).
Tetra fishes had a distinctive J-shaped, expandable saccular organ in the stomach, characterized by a single layer of columnar epithelium in the gastric mucosa.The J-shaped stomach is typical of carnivorous and omnivorous fish and enables efficient digestion of large prey, while the U-shaped stomach of herbivorous fish allows for prolonged processing of plant matter, reflecting their respective feeding behaviors (Gerking, 1994).Observed abundant neutral mucins at the apical surface of the epithelial cells are serving a protective role against autodigestion processes induced by acidic secretions from gastric glands (Bellinate et al., 2023;Cao & Wang, 2009;Faccioli et al., 2014).Gastric glands were present in the cardiac and fundic regions, playing a crucial role in prey digestion (Albrecht et al., 2001;Cao & Wang, 2009;Faccioli et al., 2014).The pyloric region exhibited unique features, including a thick smooth muscle layer, columnar cells with positive PAS staining in their apical portion, and the absence of gastric glands.
The well-developed circular smooth muscle layer in the pyloric region facilitates the mechanical force necessary to propel digested food into the intestine.In some fishes, such as mullet and trout, extreme thickening of the pyloric region resembles a gizzard, reminiscent of the avian organ used for grinding or triturating food (Wilson & Castro, 2010).
The primary function of the intestine is to finalize digestive processes initiated in the mouth and facilitate nutrient absorption (Moraes & Almeida, 2020;Wilson & Castro, 2010).Herein, the tetra fishes exhibited intestines with four main loops and pyloric caeca located in the anterior section after the pyloric stomach.Like observations in salmon species (Løkka et al., 2013) and cardinal tetra ( Rincón et al., 2023), the tetra fishes have deeply folded intestines.
In addition, as reported by Cho et al. (2023) for Pseudopleuronectes yokohamae, the thickest muscular layer and greatest perimeter and area of the lumen were observed in the posterior segment of the intestine.The intestinal lining consists of a single layer of columnar epithelium with enterocytes possessing a well-developed brush border and goblet cells.The brush border's reactivity to PAS staining, along with the presence of glycocalyx containing neutral glycoconjugates, suggests a role in emulsifying food into chyme and aiding nutrient absorption (Bakke et al., 2010;Matheus et al., 2021;Murray et al., 1996).The alkaline phosphatase presence further supports these functions.
Goblet cells were more abundant in the posterior segment of the intestine in all three tetra fishes.The mucous secretions from these cells are important in protecting the intestinal epithelium and facilitating the smooth passage of food (Moraes & Almeida, 2020).
While the specific distribution of goblet cells between the anterior and posterior sections remains unclear, their presence in both areas suggests a dual function in lubrication, aiding the movement of fecal contents and providing protection against chemical substances in the intestinal lumen, including antigens, toxins, and digestive enzymes (Wilson & Castro, 2010).This distribution pattern aligns with observations in various species across different genera, where a higher abundance of goblet cells in the posterior region is often associated with the process of defecation (Cho et al., 2023;Faccioli et al., 2014;Germano et al., 2014;Matheus et al., 2021;Rincón et al., 2023).
However, exceptions exist, such as in Leporinus species and farmed species, where a greater abundance of goblet cells was noted in the anterior intestine, potentially linked to neutralizing stomach acids (Albrecht et al., 2001;Bellinate et al., 2023).In contrast, Salmo salar showed no significant differences in goblet cell abundance between intestinal segments (Løkka et al., 2013).
Herein, this study provides a detailed overview of the digestive systems of native tetra fishes and their feeding habits.Despite being omnivores, three tetra species exhibited differences in anatomy and diet.Bryconamericus ikaa, with a smaller body size and shorter intestines, consumed more animal food items and had fewer pyloric caeca.By contrast, Psalidodon species had more pyloric caeca and a balanced diet of animal and plant materials.However, all species primarily assimilated animal food into their tissues.Histologically, their digestive tracts followed a standard vertebrate plan, with variations observed in muscular layer thickness, lumen perimeter, and goblet cell numbers among different segments.These findings provide foundational insights into the digestive systems of these species, facilitating further exploration of fish feeding strategies.Like this, the study underscores the importance of ecological and morphological data in understanding the phenotypic plasticity of omnivorous consumers with implications for species coexistence.
cies-Psalidodon bifasciatus (Garavello & Sampaio 2010), Psalidodon aff.gymnodontus Eigenmann 1911, and Bryconamericus ikaa Casciotta, Almirón & Azpelicueta 2004-from headwater streams in the Lower Iguaçu River basin (= Iguassu), Southern Brazil.The Iguaçu River basin stands out as an ecological region renowned for its uniqueness biodiversity, high endemism, and pivotal role in supporting various ecosystem types.Notably, B. ikaa and P. aff.gymnodontus are endemic to the Iguaçu river basin, underlining the importance of understanding their natural history in light of increasing anthropogenic pressures 25°9′10.25″ S, 53°16′41.86″W; S2: 25°6′7.17″S, 53°18′42.25″W) in July and December 2017 using electrofishing with three passes of 40 min.During each fieldwork we anesthetized the specimens in eugenol (Eugenol, 2 drops per litter; American Veterinary Medical Association, 2001; Javahery et al., 2012).Afterwards, we identified T A X O N O M Y C L A S S I F I C A T I O N from the MixSIAR package (Stock & Semmens, 2016).Mixing models provide robust quantitative estimates of different diet contributions (Nielsen F I G U R E 1 A schematic drawing of the general structure of the digestive tract of tetra fishes analyzed in this study, indicating the regions used in histological analysis.The drawing is based on a specimen of Psalidodon bifasciatus.Black scale bar: 1 cm.Illustrator: Presti, P. ) and doubled the mean discrimination factor and the variability estimate (SD) by the propagation of error (√ (2.SD2)), yielding the values 2.6 ± 0.42‰ for C, and 5.8 ± 0.45‰ for N. We fit the model with a Markov chain Monte Carlo sampling with the number of chains = 3, chain length = 100,000, burn-in = 50,000, thin = 50, and model 4 (Resid*Process) error structure(Stock & Semmens, 2016).We examined the model convergence with diagnostic tests (Gelmin-Rubin, Heidelberger-Welch, and Geweke) and trace plots.
sues of tetra fishes, inferred from the stable isotope mixing model, F I G U R E 2 The relative contribution (%) of main food items consumed and assimilated by tetra fishes in each sampled stream (S1 and S2).(a, b) Proportions of consumed food items in diet were estimated by volume from stomach content analysis.(c, d) The assimilated diet contributions were estimated using a MixSIAR Bayesian mixing model.SOM denotes sedimentary organic matter.Species: Bryconamericus ikaa (Bik), Psalidodon bifasciatus (Pbi), and P. aff.gymnodontus (Pgy).
The significant (p < .05)effects are given in bold.

F
Light microscopic level showing the transversal sections of cardiac (a-d), fundic (b-e), and pyloric (c-f) regions of stomach in Psalidodon bifasciatus using Masson's trichrome staining (a-c) and PAS + AB (d-f) reactions.(d-e) Cardiac and fundic regions of stomach with PAS+ cells in their apical portion with gastric glands (*).The pyloric region of stomach with simple epithelium PAS+ in their apical portion, without gastric glands, and thick circular inner of muscularis.columnar epithelium (ep), submucosa (sm), circular inner of muscularis (cm), longitudinal outer of muscularis (lm), serosa (se), and blood vessel (bv).